JPWO2022211791A5 - - Google Patents

Description

The term "oligonucleotide", as used herein to refer to primers and probes of the present application, is defined as a nucleic acid molecule comprised of two or more ribo- or deoxyribonucleotides, preferably more than three ribo- or deoxyribonucleotides. The exact size of the oligonucleotide will depend on various factors and on the particular application for which the oligonucleotide is to be used. The term "probe", as used herein, refers to an oligonucleotide, polynucleotide or nucleic acid, either RNA or DNA, whether naturally occurring in a purified restriction enzyme digest or produced synthetically, that is capable of annealing or specifically hybridizing to a sequence complementary to the probe. Probes can be either single-stranded or double-stranded. The exact length of the probe will depend on many factors, including temperature, source of the probe and method of use. For example, for diagnostic applications, depending on the complexity of the target sequence, an oligonucleotide probe typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.

The "subunits" of human or animal FVIII are herein the heavy and light chains of the protein. The heavy chain of FVIII contains three domains, A1, A2 and B. The light chain of FVIII also contains three domains, A3, C1 and C2.

Treatment can take the form of a single intravenous dose of mhFVIII , or regular or continuous administration over an extended period of time, as needed. Alternatively, therapeutic mhFVIII can be administered subcutaneously or orally in one or several doses of liposomes at various time intervals.

The dosage of the viral vector expressing mhFVIII depends on the mode of administration, the disease or condition being treated, the condition of the individual subject, the particular viral vector, and the gene being delivered, and can be determined in a routine manner. An exemplary dose to achieve a therapeutic effect is a viral titer of at least about 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ , 10 ¹² , 10 ¹³ , 10 ¹⁴ , 10 ¹⁵ transducing units or more, preferably about 10 ⁸ to 10 ¹³ transducing units, and even more preferably 10 ¹² transducing units. The polynucleotide encoding the mtFVIII of the present application may be administered as a component of a DNA molecule having appropriate regulatory elements for expression in target cells. The polynucleotide encoding the mhFVIII of the present application may be administered as a component of a viral plasmid or viral particle, e.g., an AAV particle. The viral particles may be administered as viral particles alone by direct in vivo delivery directly into the portal vein of a subject in need thereof, or as an ex vivo treatment involving transduction of cells with the viral particles ex vivo, followed by introduction of the in vivo transduced cells back into the subject.

A polynucleotide encoding mhFVIII can be used as a single chain molecule containing both heavy and light chain portions, or split into two or multiple molecules (e.g., heavy and light chains) in multiple independent viral or non-viral vectors for delivery to host cells of a patient.

Example 2 Construction, Expression and Functional Activity of Hybrid Human/Canine Factor VIII Mutants To evaluate the functional activity of the mutant h F VIII of the present application by comparing mutant hybrid human/canine FVIII consisting of mutant h F VIII heavy chain (hHC) and canine FVIII light chain (cLC), a series of B domain-less FVIII constructs were prepared and expressed in HEK 293T cells, as shown in Figures 11A and 11B.

In another aspect, the functional activity of the rAAV vector expressing the mutant hF VIII of the present application was compared with that of the rAAV vector expressing the parent hBDDF8.In this case, a series of rAAV expressing hBDDF8 protein and modified hBDDF8 protein was constructed and produced.The obtained rAAV was used to infect HuH7 cells, and the hF VIII activity in the infected cells was analyzed.

In this case, a series of rAAVs expressing mutant hF VIII of the first application were constructed in which the CAG promoter in hBDDF8 was replaced with the human TTR promoter. Briefly, pANG-CAG-hBDDF8 was digested with SnaBI and MluI. A DNA fragment encoding the TTR promoter and intron was chemically synthesized and used to replace the CAG promoter region in pANG-CAG-hBDDF8 by Gibson assembly.

Claims (20)

A modified human factor VIII (mhFVIII) protein comprising one or more amino acid substitutions at positions selected from the group consisting of A20, T21, F57, L69 , L178, R199, H212, I215, R269, I310, L318, S332, R378 and I661 of SEQ ID NO:3. 2. The m hFVIII protein of claim 1, comprising two or more amino acid substitutions at positions selected from the group consisting of A20, T21, F57, L69, L178, R199, H212, I215, R269, I310, L318, S332, R378 and I661 of SEQ ID NO:3. 2. The mhFVIII protein of claim 1, comprising two or more amino acid substitutions at positions selected from the group consisting of T21 and L178 of SEQ ID NO:3. 2. The mhFVIII protein of claim 1, comprising one or more amino acid substitutions selected from the group consisting of A20K, T21I, T21V, F57L, L69V, L178F, R199K, H212Q, I215V, R269K, I310V, L318F, S332P, R378S and I661V of SEQ ID NO:3, wherein the positions of the substitutions represent the positions of the amino acid residues of SEQ ID NO:3. 5. The m hFVIII protein of claim 4 , comprising two or more amino acid substitutions selected from the group consisting of A20K, T21I, T21V, F57L, L69V , L178F , R199K, H212Q, I215V, R269K, I310V, L318F, S332P , R378S and I661V . 5. The mhFVIII protein of claim 4, comprising one or more amino acid substitutions selected from the group consisting of T21I, T21V and L178F. The mhFVIII protein of claim 1 , comprising a truncated B domain. (1) The A1 and A2 domains of wild-type hFVIII or mhFVIII, and
( 2) A3, C1 and C2 domains of FVIII from non-human species
The mhFVIII protein of claim 1 , comprising : 9. The mhFVIII protein of claim 8 , wherein the A3, C1 and C2 domains are derived from canine factor VIII. 2. An isolated polynucleotide encoding the mhFVIII protein of claim 1 . An expression vector comprising the polynucleotide of claim 10 . The expression vector of claim 11 which is a viral vector. The expression vector of claim 12 , wherein the viral vector is an AAV vector. A host cell comprising the expression vector of claim 11 . (1) The expression vector according to claim 11 ; and
( 2) A pharmaceutical composition comprising a pharma- ceutically acceptable carrier. A method for treating a subject having factor VIII deficiency, comprising administering to said subject an effective amount of the pharmaceutical composition of claim 15. A20K, A20L, A20V, A20E, A20S, A20I, A20T, T21V, T21I, F57L, F57Y, F57S, F57P, l69V, L69I, I80V, I80T, I80L, I80M, I80Q, I80E, I80R, L178F, L178M, L178S, R199K, H212Q, I215V, R269K, R269Q, R269N, R269G, I310V, I310A, I31 1. A modified human factor VIII (mhFVIII) protein comprising two or more amino acid substitutions selected from the group consisting of: L318H, L318F, L318S, L318V, I318H, L318S, L318T, L318M, S332P, S332L, R378S, R378N, R378K, R378T, R378I, I610M and I661V, wherein the positions of said substitutions represent the positions of the amino acid residues in SEQ ID NO:3. A monomeric polynucleotide encoding the mhFVIII protein of claim 17. An expression vector comprising the polynucleotide of claim 18. A method for treating a subject having factor VIII deficiency, comprising administering to the subject an effective amount of the expression vector described in claim 19.